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Objectives To describe the propensity of carbapenem-resistant Pseudomonas aeruginosa to spread within a hospital critical care setting. Methods The study was conducted in a 700-bed tertiary centre in Cologne, Germany. P. aeruginosa resistant to piperacillin, ceftazidime, cefepime, imipenem, meropenem and ciprofloxacin, isolated from clinical and screening specimens from four critical care units from 2015 to 2020 were analysed. Genotyping was carried out by WGS (Illumina and MinION). MLST, core genome MLST (cgMLST) and resistome analysis was performed and merged with epidemiological data. Results Fifty-five out of 79 non-duplicate P. aeruginosa isolates were available, of which 20 were carbapenemase producers as follows: blaVIM-1 (n = 1), blaVIM-2 (n = 17), blaVIM-4 (n = 1), and blaNDM-1/blaGES-5 (n = 1). Forty-two of 55 isolates were hospital-acquired. cgMLST revealed three clusters: Cluster 1 (n = 15, ST111, blaVIM-2, recovered between 2015 and 2020); Cluster 2 (n = 4, ST970, carbapenemase negative); and Cluster 3 (n = 2, ST357, carbapenemase negative). The blaVIM-2 gene of Cluster 1 was integrated on the chromosome in a class 1 integron (type In59). Using conventional epidemiology, we were only able to confirm two patient-to-patient transmissions and one room-to-patient transmission on three different ICUs within Cluster 1. Isolates from Cluster 2 represented an outbreak occurring in 2019. Conclusions These data give insight into the epidemiology of carbapenem-resistant P. aeruginosa. Transmission dynamics differed between carbapenemase- and non-carbapenemase-producing isolates. A continuous acquisition of clonally related ST111 VIM-2 P. aeruginosa, being the main carbapenemase-producing strain, was observed over the whole study period, as well as an overall higher genomic diversity among non-carbapenemase-producing P. aeruginosa.

In limelight of the ongoing pandemic SARS-CoV-2 testing is critical for the diagnosis of infected patients, contact-tracing and mitigating the transmission. Diagnostic laboratories are expected to provide appropriate testing with maximum accuracy. Real-time reverse transcriptase PCR (RT-PCR) is the diagnostic standard yet many commercial diagnostic kits have become available. However, only a handful of studies have reviewed their performance in clinical settings. The aim of this study was to compare the performance of the overall analytical matrix including the extraction kit (BD MAX, Promega, Qiagen), the PCR instrument (Agilent Mx3005P, BD MAX, Qiagen Rotor-Gene, Roche Cobas z 480) and the RT-PCR assay (Altona Diagnostics, CerTest Biotec, R-Biopharm AG) using predefined samples from proficiency testing organizers.The greatest difference of the Ct values between the matrices was 9 cycles. One borderline sample could not be detected by 3 out of 12 analytical matrices and yielded a false negative result. We therefore conclude that diagnostic laboratories should take the complete analytical matrix in addition to the performance values published by the manufacturer for a respective RT-PCR kit into account. With limited resources laboratories have to validate a wide range of kits to determine appropriate analytical matrices for detecting SARS-CoV-2 reliably. The interpretation of clinical results has to be adapted accordingly.

Background A. baumannii is a common nosocomial pathogen known for its high transmission potential. A high rate of carbapenem-susceptible Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB)-complex in clinical specimens led to the implementation of a pathogen-based surveillance on a 32-bed surgical intensive care unit (SICU) in a German tertiary care centre. Methods Between April 2017 and March 2018, ACB-complex isolates with an epidemiological link to the SICU were further assessed. Identification to the species level was carried out using a multiplex PCR targeting the gyrB gene, followed by RAPD, PFGE (ApaI) and whole genome sequencing (WGS, core genome MLST, SeqSphere+ software, Ridom). Additional infection prevention and control (IPC) measures were introduced as follows: epidemiological investigations, hand hygiene training, additional terminal cleaning and disinfection incl. UV-light, screening for carbapenem-susceptible A. baumannii and environmental sampling. Hospital-acquired infections were classified according to the CDC definitions. Results Fourty four patients were colonized/infected with one or two (different) carbapenem-susceptible ACB-complex isolates. Fourty three out of 48 isolates were classified as hospital-acquired (detection on or after 3rd day of admission). Nearly all isolates were identified as A. baumannii, only four as A. pittii. Twelve patients developed A. baumannii infections. Genotyping revealed two pulsotype clusters, which were confirmed to be cgMLST clonal cluster type 1770 (n = 8 patients) and type 1769 (n = 12 patients) by WGS. All other isolates were distinct from each other. Nearly all transmission events of the two clonal clusters were confirmed by conventional epidemiology. Transmissions stopped after a period of several months. Environmental sampling revealed a relevant dissemination of A. baumannii, but only a few isolates corresponded to clinical strains. Introduction of the additional screening revealed a significantly earlier detection of carbapenem-susceptible A. baumannii during hospitalization. Conclusions A molecular and infection surveillance of ACB-complex based on identification to the species level, classic epidemiology and genotyping revealed simultaneously occurring independent transmission events and clusters of hospital-acquired A. baumannii. This underlines the importance of such an extensive surveillance methodology in IPC programmes also for carbapenem-susceptible A. baumannii.

Background Pseudomonas aeruginosa is a common pathogen causing hospital-acquired infections. Carbapenem resistance in P. aeruginosa is either mediated via a combination of efflux pumps, AmpC overexpression, and porin loss, or through an acquired carbapenemase. Carbapenemase-producing P. aeruginosa (CPPA) strains are known to cause outbreaks and harbour a reservoir of mobile antibiotic resistance genes, however, few molecular surveillance data is available. The aim of this study was to analyse the prevalence and epidemiology of CPPA in three German medical centres from 2015 to 2017. Methods Identification and susceptibility testing were performed with VITEK 2 system. P. aeruginosa non-susceptible to piperacillin, ceftazidime, cefepime, imipenem, meropenem and ciprofloxacin (4MRGN according to the German classification guideline) isolated from 2015 to 2017 were analysed. A two-step algorithm to detect carbapenemases was performed: phenotypic tests (EDTA- and cloxacillin-combined disk tests) followed by PCR, Sanger sequencing, and eventually whole genome sequencing. CPPA isolates were further genotyped by RAPD and PFGE. In-hospital transmission was investigated using conventional epidemiology. Results Sixty two P. aeruginosa isolates were available for further analysis, of which 21 were CPPA as follows: blaVIM-1 (n = 2), blaVIM-2 (n = 17), blaNDM-1/blaGES-5 (n = 1) and the newly described blaIMP-82 (n = 1). CPPA were mostly hospital-acquired (71.4%) and isolated on intensive care units (66.7%). All (except one) were from the tertiary care centre. PFGE typing revealed one large cluster of VIM-2-producing CPPA containing 13 isolates. However, using conventional epidemiology, we were only able to confirm three patient-to-patient transmissions, and one room-to-patient transmission, on several intensive care units. Conclusions These data give insight into the epidemiology of CPPA in three centres in Germany over a period of 3 years. Carbapenemases are a relevant resistance mechanism in 4MRGN-P. aeruginosa, illustrated by genetically related VIM-2-producing strains that seem to be endemic in this region. Our data suggest that infection control measures should especially focus on controlling transmission on the ICU and support the need for a local molecular surveillance system.

Clostridium (Clostridioides) difficile is a major cause of nosocomial diarrhoea. A first inter-laboratory ring trial was performed in four European countries to evaluate the genotyping and antibiotic susceptibility testing (AST) accuracy. Six C. difficile isolates representing the epidemiologic important ribotypes (RT), RT001, RT002, RT010, RT014, RT027, and RT078 were blinded and send to 21 participating laboratories. Participants tested the samples with their genotyping and AST methods in use for concordance with reference. A total of 21 genotyping- and 14 antimicrobial susceptibility data sets were obtained. Ribotyping (11 participants) correctly identified most RTs (median 91% concordance rate) except for RT002, which was misidentified in 4/11 reports. However, this isolate was correctly asserted to RT002 after an update of a publicly available ribotyping database. Multilocus sequence typing, surface layer sequence typing, DNA microarray based genotyping, and whole genome sequencing, which were used by 1–3 participants, identified all six isolates correctly. AST was done by epsilometry by the participants and compared to agar dilution data determined by the coordinating reference centre. Susceptibilities against metronidazole, moxifloxacin, and vancomycin were correctly identified in 235 of 237 cases and in accordance to agar dilution as the gold standard. Genotyping of the C. difficile test strains revealed a remarkable high concordance on the level of ribotypes with a wide variety of methods. Epsilometry appears to be a reliable method for AST of C. difficile isolates in routine clinical microbiology laboratories.

We present findings describing the epidemiology of non-severe acute respiratory syndrome human coronavirus-associated influenza-like illness from a population-based active follow-up study in four different regions of Peru. In 2010, the prevalence of infections by human coronaviruses 229E, OC43, NL63, or HKU1 was 6.4% in participants with influenza-like illness who tested negative for influenza viruses. Ten of 11 human coronavirus infections were identified in the fall–winter season. Human coronaviruses are present in different regions of Peru and are relatively frequently associated with influenza-like illness in Peru.

The human bocavirus was first detected in 2005 and since then has been found in both respiratory secretions from patients with airway infections and in stool samples from patients with gastroenteritis. Meanwhile, four different genotypes have been identified that most likely derive from recombination events. Although the modified Koch’s postulates have not yet been fulfilled completely, owing to the lack of an animal model or a simple cell culture system, there is increasing evidence that the human bocaviruses are serious participants in infectious diseases of the respiratory and the GI tracts. This article reviews the current status of the clinical features of human bocaviruses and provides an overview of the latest findings concerning the biology, phylogeny, epidemiology and diagnostic tools related to human bocaviruses. Furthermore, it discusses the potential pathogenicity of human bocavirus, as well as its persistence and reactivation in hosts.

A.baumannii (AB) colonisation and infection is frequent in burn patients and multiple outbreaks especially with multidrug resistant strains are described in burn units as well as in other intensive care units. The understanding of the transmission routes of AB in health care settings is eminent to control outbreaks and prevent transmission. Burn patients are suspected to spread high amounts of pathogens.

The immunoregulatory cytokine IL-10 suppresses T-cell immunity. The complementary question, whether IL-10 is also involved in limiting the collateral damage of vigorous T cell responses, has not been addressed in detail. Here, we report that the particularly strong virus-specific immune response during acute primary infection with the lymphocytic choriomeningitis virus (LCMV) in mice is significantly further increased in Il10-deficient mice, particularly regarding frequencies and cytotoxic activity of CD8(+) T cells. This increase results in exacerbating immunopathology in select organs, ranging from transient local swelling to an increased risk for mortality. Remarkably, LCMV-induced, T cell-mediated hepatitis is not affected by endogenous Il10. The alleviating effect of Il10 on LCMV-induced immunopathology was found to be operative in delayed-type hypersensitivity footpad-swelling reaction and in debilitating meningitis in mice of both the C57BL/6 and BALB/c strains. These strains are prototypic counterpoles for genetically imprinted type 1-biased versus type 2-biased T cell-mediated immune responses against various infectious pathogens. However, during acute LCMV infection, neither systemic cytokine patterns nor the impact of Il10 on LCMV-induced immunopathology differed conspicuously between these two strains of mice. This study documents a physiological role of Il10 in the regulation of a balanced T-cell response limiting immunopathological damage.

We report two confirmed cases of usual interstitial pneumonia (UIP) associated with infection of the human bocavirus (HBoV). In one case HBoV was identified in the bronchoalveolar lavage (BAL) during an acute exacerbation as well as post mortem in different tissues giving raise to the hypothesis that HBoV infections trigger UIP or could be a causative agent and be a systemic component in UIP. In the other case, the UIP was confirmed by radiological methods and HBoV was detected in the BAL during an acute exacerbation. Both cases give raise to the hypothesis that HBoV could be a causative agent of UIP or could contribute to its development and/or acute exacerbations.

Human bocavirus (HBoV), discovered in 2005, can cause respiratory disease or no symptoms at all. We confi rmed HBoV infection in an 8-month-old girl with hypoxia, respiratory distress, wheezing, cough, and fever. This case demonstrates that lower respiratory tract infection caused by HBoV can lead to severe and life-threatening disease.

Background Cause for gastroenteritis range from viral, bacterial to parasitic pathogens. Rapid Multiplexing techniques like ProGastro_SSCS and xTAG_GPP can detect broad panels of pathogens simultaneously. We performed a field test with a total number of 347 stool samples from adult hospitalized patients that were tested with the Luminex xTAG GPP assay; of the 157 samples positively tested for at least one pathogen by xTAG GPP a total number of 30 samples was retested with the ProGastro SSCS assay. Assays were compared to standard routine diagnostics. Findings Multiplexing significantly reduced the time to the initial identification of a pathogen. Moreover, multiplexing detected pathogens for which a diagnostic assays was not requested by the physician and thus may be an important tool for avoiding nosocomial outbreaks. Conclusion This first frontline approach with these assays approves their utility compared to conventional microbiological methods.

Human bocavirus is the second autonomous human parvovirus with assumed pathogenic potential. Other parvoviruses are known to persist and even integrate into the host genome, eventually contributing to the multi-step development of cancer. Human bocavirus also persists in an unknown percentage of clinically asymptomatic patients in addition to those with primary infection. The aim of the present study was to analyze the role of Human bocavirus in lung and colorectal cancers. Therefore, formalin-fixed, paraffin-embedded, archived tumor samples were screened for Human bocavirus DNA by PCR, Southern blotting, and sequencing. Positive tissues were further subjected to fluorescence in situ hybridization analysis to specifically detect human bocavirus DNA in the infected cells. In total, 11 of the 60 (18.3%) lung and 9 of the 44 (20.5%) colorectal tumors tested positive for human bocavirus DNA by PCR and were confirmed by sequencing and fluorescence in situ hybridization analysis. Thus, human bocavirus DNA is present in the nuclei of infected cells, in either single or multiple copies, and appears to form concatemers. The occurrence of these human bocavirus DNA structures supports the existence of the postulated σ- or rolling-hairpin replication mechanism. Moreover, the fluorescence in situ hybridization patterns inspired the hypothesis that human bocavirus DNA either persists as cccDNA or is integrated into the host genome. This finding suggests that this virus may indirectly contribute to the development of some colorectal and lung cancers, as do other DNA viruses, such as the human hepatitis B virus, or may play an active role in cancer by interacting with the host genome.

A case of severe diarrhoea associated with synergistic human bocavirus type 1 (HBoV) and human herpes virus type 6 (HHV6) is reported. The case supports the hypotheses that HBoV infection under clinical conditions may depend on helper viruses, or that HBoV replicates by a mechanism that is atypical for parvoviruses, or that HBoV infection can be specifically treated with cidofovir.